1. Field of the Invention
The present invention relates generally to the fields of botany and molecular biology. More specifically, the invention relates to plant transformation using Agrobacterium-mediated gene transfer into the stolon of plants.
2. Description of the Related Art
Forage and turf grasses are the backbone of sustainable agriculture and contribute extensively to the world economy. On a worldwide basis, grassland acreage is estimated to be twice that of cropland (Jauhar, 1993). Along with cereal crops, these monocotyledonous (monocot) species have been considered recalcitrant for genetic transformation (Ke et al., 2001; Sahrawat et al., 2003; Spangenberg et al., 1998; Vasil, 1994). Transgenic monocot plants were first obtained by direct gene transfer to protoplasts, then by biolistic transformation, and in more recent years by Agrobacterium-mediated transformation (Cheng et al., 2004; Janakiraman et al., 2002; Spangenberg et al., 1998; Wang et al., 2001).
While direct gene transfer to protoplasts remain useful for transient expression assay, biolistic and Agrobacterium-mediated transformation are the two major methods for generating transgenic plants in monocots (Cheng et al., 2004; Janakiraman et al., 2002; Wang et al., 2001). Agrobacterium-mediated transformation has received more attention in recent years, because it has the advantage of allowing for the stable integration of a defined DNA segment into the plant genome and generally results in a lower copy number, fewer rearrangements and an improved stability of expression over generations than the free DNA delivery methods (Dai et al., 2001; Hu et al., 2003).
Callus culture has been an inevitable step in monocot and other plant transformation protocols. In many transformation protocols, calluses were used as direct target for microprojectile bombardment or for Agrobacterium infection (Cheng et al., 2003; Cho et al., 2001; Hartman et al., 1994; Li and Qu, 2004; Sallaud et al., 2003; Spangenberg et al., 1998; Spangenberg et al., 1995; Vasil et al., 1992; Wan and Lemaux, 1994; Wang et al., 2004; Wang and Ge, 2005). In other protocols, freshly isolated immature embryos or shortly precultured embryos were used as target for microprojectile bombardment or for Agrobacterium infection (Aldemita and Hodges, 1996; Frame et al., 2002; Hu et al., 2003; Huber et al., 2002; Popelka and Altpeter, 2003; Tingay et al., 1997; Wan and Lemaux, 1994; Zhao et al., 2000), and calluses were later induced from the bombarded or infected embryos. Callus induction and plant regeneration from the induced callus is not only time consuming and laborious, but also causes somaclonal variation (Choi et al., 2000; Goldman et al., 2004; Spangenberg et al., 1998). Thus, there remains a need for improved methods of Agrobacterium-based transformation.